6-methyl-19-nor steroids and intermediates therefor



United States Patent Ofihce 3,314,944 Patented Apr. 18, 1967 Thisinvention relates to and has as its object the provision of newphysiologically active steroids, methods for preparing the same, and newintermediates useful in said preparation.

The final products of this invention can be represented by the formulaICH3 9 wherein R and R each represents hydrogen; and together R and R iswherein P represents hydrogen, lower alkyl, halo lower alkyl, monocycliccycloalkyl, monocyclic cycloalkyl lower alkyl, monocyclic aryl,monocyclic aryl lower alkyl, monocyclic heterocyclic or monocyclicheterocyclic lower alkyl; Q is lower alkyl, halo lower alkyl, monoeycliccycloalkyl, monocyclic cycloalkyl lower alkyl, monocyclic aryl,monocyclic aryl lower alkyl, monocyclic heterocyclie or monocyclicheterocyclic lower alkyl; or together with carbon to which they arejoined P and Q is a monocyclic cycloalkyl or monocyclic heterocyclicradical.

The final products of this invention are physiologically activesubstances which possess progestational activity and hence, can be usedin lieu of known progestational agents, such as progesterone, in thetreatment of habitual abortion. For this purpose, they can beadministered in the same manner as progesterone, for example, the dosagebeing adjusted for the relative potency of the particular steroid. Thecompounds of this invention can also be administered perorally in theform of tablets. Moreover, it has surprisingly been found that thecompounds of this invention are many times more active than are thecorresponding lO-methylated derivatives.

In the most preferable embodiment of this invention, P is lower alkyland Q is selected from the group consisting of lower alkyl (e.g.,methyl) and monocyclic aryl (e.g., phenyl).

The final products of this invention are prepared according to the novelprocesses of this invention, which may be represented by the followingequations and Q are as hereinbefore defined.

wherein R, P

CH3 CH;

ozoggfiiixioc CH3 CH3 H O Q CHz (V) (VII) Z =6,7-dihydro (VI) (VI) Z=Adehydro 1604,1711 dihydroxyprogesterone 16,17 acetophenonide may beprepared according to the procedures and disclosures of copendin'gapplication Ser. No. 99,732, filed Mar. 31, 1961, in the names ofPatrick A. Diassi 3 and Josef Fried and application Ser. No. 830,467,filed July 30, 1959, in the name of Josef Fried.

In the first step of the process of this invention, the startingmaterial (Compounds A and B) is subjected to the action of amicroorganism selected from the group consisting of Corticiummicrosclerotia, Corticium sasalkii, Corticium praticola, Hypochnussasakii and Pellz'cula'ria filamentosa or to the action of the enzymesthereof under oxidizing and preferably aerobic conditions.

To prepare the 19-hydroxy compounds of this invention (Compounds C), thestarting materials (Compounds A and B) may first be subjected to theaction of the enzymes selected from the group consisting of Corticiummicrosclerotia NRRL-2727, Hypoclmus sasakii ATCC- 13290, Pelliculariafilamentosa ATCC-13289, Corticium pratz'cola NRRL-2724, and Corticiumsasakii NRRL- 2705 under oxidizing conditions. This oxidation can bestbe effected either by including the starting material in an aerobicculture in the desired microorganism or by bringing together in anaqueous medium the compounds, air and enzymes of non-proliferating cellsof the microorganism.

In general, the conditions of culturing the microorganism for thepurposes of this invention are (except for the inclusion of the startingmaterials to be converted), the same as those of culturing various othermicroorganisms for the production of antibiotics, vitamin B12, and otherlike substances. The microorganism is grown aerobically in contact with(in or on) suitable fermentation medium. A suitable medium essentiallycomprises a source of carbon and energy. The .latter may be acarbohydrate, for example, molasses, glucose, maltose, starch ordextrin, a fatty acid, a fat and/ or the compound itself. Preferably,however, the medium includes an assimilable source of carbon and energyin addition to the steroid. Among the fats utilizable for the purpose ofthis invention are lard oil, soybean oil, linseed oil, cottonseed oil,peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palmoil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin,triolein and trilaurin. Among the fatty acids utilizable for the purposeof this invention are stearic acid, palmitic acid, oleic acid, linoleicacid and myristic acid.

The source of nitrogenous factors utilizable for the purposes of thisinvention may be organic (e.g., soybean meal, cornsteep liquor, yeastextract, meat extract and/ or distillers solubles) or synthetic (i.e.,composes of simple synthesizable organic or inorganic compounds, such asammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thecompound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of the concentration of the compoundin the culture is about 0.01% to about 0.2%. The culture period (orrather the time of subjecting the compound to the action of the enzyme)may vary considerably, the range of about eight to ninety-six hoursbeing feasible, but not limiting.

The l6oc,17cck6'tal or acetal derivatives of the6-methyll9-hydroxyprogesterone (Compounds C) may then be oxidized as bytreatment with chromic anhydride in sulfuric acid, to yield thel6a,17a-ketal or acetal derivatives of 6-methyl-19-oxo-progesterone(Compounds D), which are also new compounds of this invention.

To obtain the 16a,l7u-ketal or acetal derivatives of 6-methyl-19-norprogesterone, the final products of this invention(Compounds E) the 160:, 17a-ketal or acetal derivatives of 6methyl-l9-hydroxyprogesterone (Compounds C) andG-methyl-19-oxoprogesterone (Compounds D) may be hydrolyzed as bytreatment with an alkali metal hydroxide, such as potassium hydroxide orsodium hydroxide.

If a ketal or acetal group other than that present in the intermediateor final compounds or starting material is desired, the respectiveroduct may be cleaved by treatment with aqueous formic acid to yield therespective 16m,170c-dihyd1'OXy intermediates. These intermediates arethen reacted with an aldehyde or ketone of the formula wherein P and Qare as hereinbefore defined. The reaction is preferably carried out bytreating a suspension or solution of the 16,17-dihydroxy steroid in thealdehyde or ketone (or an organic solvent and the aldehyde or ketone, ifthe aldehyde or ketone is a solid) with an acid catalyst (e.g.,perchloric acid, p-toluenesulfonic acid, hydrochloric acid, etc.),neutralizing the acid and recovering the acetal or ketal derivativeformed. Suitable aldehyde and ketone reactants include those set forthin US. Patent 3,077,471, issued Feb. 12, 1963.

The following examples are illustrative of the invention (alltemperatures being in centigrade):

EXAMPLE 1 6 ot-methyl-l 6 oc,] 7a,] 9-tr'ihydroxyprogesterone16,17-acetonide Surface growth from ten to fourteen-day-old agar slantcultures of Coriicium microsclerotfa (NRRL 2727 Northern RegionalResearch Laboratories, Peoria, Illinois) the slant containing asterilized nutrient medium A:

Baby oatmeal g 20.0 Tomato paste g 20.0 Agar g 15.0 Tap water l 1.0

is suspended in 5.0 ml. of a 0.01% Duponal (wetting agent) aqueoussolution. One ml. portions of the suspension from two slants are used toinoculate six 250 ml. conical flasks, each containing 50 ml. of thefollowing sterilized medium B:

G. Glucose 30.0 Soy bean meal 20.0 Soy bean oil 2.2 CaCO 2.5

Distilled water to 1 liter. 1

After four days incubation at 25 on a rotary shaker operating at 280cycles per minute with a stroke of 2", 10% (v.:v.) transfers are made to50 conical flasks each containing 50 ml. of the following sterilizedmedium C adjusted to pH 7.0:

. G. Corn steep liquor 6.0 NH H PO 3.0 Yeast extract 2.5 Dextrose 10.0CaCO 2.5

Distilled water to 1 liter.

After one day of incubation under the conditions described for medium B,6a-methyl-16a,l7a-dihydroxyprogesterone is added to each flask as 0.25ml. of a sterile solution containing 10.0 mg. of the steroid inN,N-dimethylformamide. A total of 500 mg. is used. After twenty-fourhours of further incubation, the contents of the flasks are pooled, theculture filtered on a Buchner funnel through a Seitz clarifying pad andwashed with Water. The combined filtrate and washings are extractedthree times with 800 ml. portions of ethyl acetate which are combined,washed twice with 1 liter portions of water, dried over sodium sulfateand evaporated to dryness, in vacuo. The residue is dissolved in 35 ml.of acetone containing 0.035 ml. of perchloric acid and left at roomtemperature for sixteen hours. The reaction mix J ture is thenneutralized with sodium bicarbonate diluted with water and extractedwith ethyl acetate. The ethyl acetate is washed with water, dried oversodium sulfate, and evaporated to dryness, in vacuo. This residue isthen dissolved in ml. of dry pyridine, 5 m1. of acetic anhydride areadded and the solution kept at room temperature for fifteen hoursprotected from moisure. After hydrolysis of the excess acetic anhydridewith ice water, the mixture is extracted several times with chloroformand the combined chloroform extracts washed successively with 2 Nhydrochloric acid, 5% sodium bicarbonate and Water, dried and evaporatedto dryness, in vacuo. The residue is dissolved in a few milliliters ofchloroform and adsorbed onto a plate (4 mm. x cm. x 30 cm.) of Woelmneutral alumina (Activity V). Development of the plate with chloroformgives three principal bands detectable by ultraviolet light at R 0.7-0.8and 0.5, respectively.

One of the bands having Rf 07-08 is eluted with ethyl acetate andevaporated to dryness, in vacuo. The residue is dissolved in 10 ml. ofmethanol, 1.0 ml. of 10% potassium carbonate is added and the mixturestirred at room temperature for sixteen hours. The solution is thenneutralized with 10% acetic acid, diluted with water, and extracted withchloroform. The chloroform is washed with water and evaporated todryness, in vacuo. The residue is plate chromatographed using Woelmneutral alumina (Activity I) as adsorbant and chloroform: ethyl acetate(10:1) as developing solvent to give a band detectable by UV. at R 0.6which on eiution with ethyl acetate, evaporation of the solvent, invacuo, and crystallization of the residue from acetone-hexane gives 60:-methyl-16a,17a,l9-trihydroxyprogesterone 16,17-acetonide.

EXAMPLE 2 605-1716lh) l-1 6 11,] 7 Ot,1 9-trz'hydroxyprogeslerone 16,17-acet0nirle (A) Five flasks of medium inoculated (10%, v.:v.) withinoculum obtained as described in Example 1. After one day of incubationas described in Example 1, 6amethyl-16a,17adihydroxyprogesterone isadded to each flask as 1.0 ml. of a solution containing 100 mg. of thesteroid as the cycloborate ester. The solution is prepared by mixing 500mg. of the steroid, 2.5 ml. of methanol, and 95 mg. of Na B O -10H Odissolved in 1.9 ml. of water in a test tube and heating at 90 in awater bath until the material is solubilized. The volume of the solutioncontaining the cycloborate ester is adjusted to 5.0 ml. with Waterbefore use. After forty-eight hours of further incubation, the contentsof the flasks are pooled and the culture filtered on a Buchner funnelthrough a Seitz clarifying pad. The filtrate is acidified to about pH3.5 by treatment with a mineral acid, such as sulfuric acid, therebyhydrolyzing the 16,17-cycloborate ester group and yielding the free16a,17u-dihydroxyfunctional groups.

(B) Following the procedure for the extraction, acetonation,acetylation, hydrolysis and isolation described in Part A of thisexample, there is obtained 60t-H16thYl-16u,17a,l9-trihydroxyprogesterone 16,17-acetonide.

EXAMPLE 3 6u-methy l-1 6 0a,] 7 a,] 9-trihydroxyprogesterone6,17-acet0nide Following the procedure of Example 1, but substituting6a-methyl-16a,l7a-dihydroxyprogesterone 16,17-acetonide for the6a-methyl-16a,17a-dihydroxyprogesterone and eliminating that part of theprocedure involving reaction with acetone and perchloric acid, there isobtained 6w methyl-le ma,19-trihydroxyprogesterone 16,17-acetonide.

6 EXAMPLE 4 6ot-methyl-19-hydroxy-16a,I 7u-(B-methyl-u-phenylmethylenedioxy -pr0gester0ne Following the procedure of Example 1, butsubstituting 6zx-methyl 16a,17a- (fl-methyl-a-phenylmethylenedioxy)-progesterone for the 60t-I116tl1Yl-160t,l7cz-dlhYdIOXYpI'Og6S- teroneand eliminating that part of the procedure involving reaction withacetone and perchloric acid, there is obtained 6ot-methyl-19-hydroxy-16ot,170t-(fi-II16thyl.-0L- phenylmethylenedioxy) -progesterone.

EXAMPLE 5 6 -mezhyl-6-dehydro-1 6 0a,] 7 0a,] 941-172 droxyprogeslerone1 6,1 7 -acetonide Following the procedure of Examples 1 and 2, butsubstituting 6methyl-6-dehydro 16a,17wdihydroxyprogesterone for6a-methyl- 16a,17a-dihydroxyprogesterone, there is obtained6-methyl-6-dehydro 16u,17a,19-trihydroxyprogesterone 16,17-acetonide.

EXAMPLE 6 6 -methyl -6-dehydr0-1 6 u,1 7a,] 9-trihyalr0xy progesterone16,17-acet0nide Following the procedure of Example 3, but substituting6-methyl 6-dehydro 1604,17ot dihydroxyprogesterone 16,17-acetonide forthe 6a-methyl 16u,17ot-dihydroxyprogesterone 16,17-acetonide, there isobtained 6-methy1 6-dehydro-16oc,17a,19-trihydroxyprogesterone16,17-acetonide.

EXAMPLE 7 6-methyl-6-dehydro-19-lzydroxy-16tx,l 7u-( fi-methyl-wphenylmethylenedioxy -pr0gester0ne Following the procedure of Examples 1and 2, but substituting 6-methyl 6-dehydro 16a,17a-dihydroxyprogesteronefor the 6a-methyl-l6a,17a-dihydroxyprogesterone and acetophenone for theacetone in the ketalization part of the experiment, there is obtained6-methyl-6-dehydro- 19-hydroxy 1604,170:(fi-methyl-u-phenylmethylenedioxy)-progesterone.

EXAMPLE 8 6methyl-6-dehydro-19-hydroxy-I 6 0a,] 7 oz- ,B-methyla-phenylmethy lenediaxy -pr0gester0ne Following the procedure of Example 3, butsubstituting 6 methyl 6dehydro-16a,17a-([3-methyl-u-phenylmethylenedioxy)-progesterone for the6u-methyl-16u,17adihydroxyprogesterone 16,17acetonide, there is obtained6 methyl 6 dehydro-19-hydroxy-16a,17a-([3-methyla-phenylmethylenedioxy)-progesterone.

EXAMPLE 9 6a-methyl-1 6a,] 7 0a,] 9-trihydroxy progesterone 16,17-acetonia'e Following the procedures of Examples 1 to 8, but substitutingany one of the following organisms: Corticium sasakii, Corticl'umpmticola, Hypochizus sasakii, and Pellicularia filamentosa for theCorticium microsclerotia, there is obtainedwmethyl-lfia,17a,19-trihydroxyprogesterone 16,17-acetonide.

EXAMPLE 10 6 a-methy l-] 6 0a,] 7 a-dimethy lmelhyl'enedioxy-I 9-oxoprogesterone a; to dryness in vacuo and the residue chromatographedon alumina (Activity V) to give on crystallization 6a-methyl- 16oz,17a-dimet'hy1methylene dioxy-1 9-oxoprogesterone'.

EXAMPLE ll 60t-mfl1yl-160L,1 70Mfi-methyLot-phenylmethylenedioxy 9-ox0progesterone Following the procedure of Example 10, but substituting 60cmethyl 16ot,17w(fi-methyl-u-phenylmethylene dioxy)19-hydroxyprogesterone for 6ot-methyl-16a,17odirnethylmethylenedioxy-19-hydroxyprogesterone, there is obtained 60methyl-16a,l7x-(fi-methyl-a-phenylmeth ylenedioxy) -19-oxoprogesterone.

EXAMPLE 12 6u-methyl-1 6a,] 7a-dimerhylmethylenedioxy-l 9-norprogesterone To a solution of 30 mg. of6u-methyl-l6a,17ot-dimethylmethylenedioxy-19-hydroxyprogesterone in 20ml. of methanol is added a solution of 1.3 g. of potassium hydroxide in10 ml. of water and the resulting solution left at room temperature foreighteen hours. The solution is then concentrated in vacuo at C. dilutedwith water and extracted with ether. The ether is washed with water,dried over sodium sulfate and evaporated to dryness in vacuo. Thicklayer plate chromatography of the residue using Activity V Woelm neutralalumina and chloroformzhexane (1:4) as solvent gives a band detectableby U.V. at Rf 0.5 which on elution with ethyl acetate andcrystallization of the residue yields606-I116thY1-1606,170tdimethylmethylenedioxy-19-norprogesterone.

EXAMPLE 13 Gu-methyZ-I 604,1 7u-dimethylmethylenedioxy-I 9-norprogesterone A solution of 200 mg. of 6a-methyl-16a,17m-dimethy1-methylenedioxy 19-oxoprogesterone in 14 ml. of methanol is added to 150ml. of 4% sodium hydroxide and the mixture warmed at 50-55 forforty-five minutes. The reaction mixture is cooled, extracted withether, the ether washed with Water, dried and evaporated to dryness.Crystallization of the residue following purification by chromatographyas described in Example 12 gives 60:- methyl 16,17adimethylmethylenedioxy 19 norprogesterone.

EXAMPLE 14 Following the procedure set forth in Example 12, butsubstituting 6a, methyl 16ot,17u 8-methyl-a-phenylmethylenedioxy) 19hydroxyprogesterone for 6u-methyl 161%,1706 dimethylmethylenedioxy 19hydroxyprogesterone, there is obtained 60: methyl-l6a,17a-(/3- methyl aphenylmethylenedioxy)-19-norprogesterone.

EXAMPLE l 6o:-methyl-I6ot,1 7a- (B-methyl-a-phenylmelhylenedioxy -19-norprogester0ne Following the procedure set forth in Example 13, butsubstituting 6a methyl 16a,17a (,8- methyla-phenylmethylenedioxy)a19-oxoprogesterone for 6a-methyl-16u,17ot-dimethylmethylenedioxy-19-oxoprogesterone, there is obtained 60:methyl 16,17a (5 methyl-a-phenylmethylenedioxy) -19-norprogesterone.

EXAMPLE 16 6-methyl-6-dehydroJ 6a,] 7a-dimezhylmethylenedioxy-1 9-0x0progesterone Following the procedure of Example 10, but substituting 6methyl 6-dehydro-16a,17a-dimethylmethylenedioxy 19 hydroxyprogesteronefor 6a-methyl-16a,17umethylenedioxy l9 hydroxyprogesterone, there isobwater.

tained 6methyl-6-dehydro-l6a,l7ot-dimethylmethylenedioxy-l9-oxoprogesterone.

Similarly, 6-methyl-6-dehydro-160:,17ot(fl-methyl-uphenylmethylenedioxy)-l9-oxoprogesterone can be preparedstarting With 6-methyl 6 dehydro-l6a,l7u-([i'- methyl aphenylmethylenedioxy) 19 hydroxyprogesterone.

EXAMPLE 17 6-methyZ-6-dehydro 16a,17 x dimelhylmethylenedioxy- 19norprogesterone and 6 methyl 6 dehydrm16a,]74x-(fi-methyl-u-phenylmethylenedioxy) 19-1101- progesterone6-melhyl-6-dehydr0-1 6 1,1 7a-dimethylmethylerredioxy-I9-n0rpr0gester0ne To a solution of 100 g. of6ot-rnethyl-16a,17u-dimethylmethylenedioxy 19 norprogesterone in 10 ml.of dioXane mg. of 2,3-dichloro-5,6-dicyanbenzoquinone are added andhydrogen chloride is bubbled through the resulting solution for thirtyseconds. After two hours at room temperature, the mixture is filteredand washed with chloroform. The combined filtrate and washings arepassed through 20 g. of Woelm neutral alumina (Activity I) and thealumina is washed with chloroform. Evaporation of the eluate in vacuoand crystallization of the residue gives6-methyl-6-dehydro-16a,l7u-dimethylmethylenedioxy-19-norprogesterone.

Similarly, starting with6a-methyl-16a,17ot-(fl-methyltat-phenylmethylenedioxy) 19norprogesterone, 6amethyl-6-dehydro 16a,17 x-(firnethyl-ot-phenyl-methylenedioxy)-19-norprogesterone is prepared.

EXAMPLE 19 6-methyZ-6-dehydr0-16aJ 7a-dihydroxy- 19-n0rpr0gester0ne Asolution of 6 methyl-6-dehydro 16a,l,7o-di-methy1- methylenedioxy 19norprogesterone in formic acid is heated at 42 for twenty-two hours. Thesolvents are removed in vacuo, the crude residue dissolved in methanoland treated under nitrogen with stirring with a 10% oxygen free solutionof potassium carbonate in After thirteen minutes at room temperature,the mixture is neutralized with glacial acetic acid and the solutionconcentrated in vacuo after the addition of Water. Extraction withchloroform followed by drying over sodium sulfate and evaporation invacuo furnishes a residue which on recrystallization from methanolfurnishes pure 6 methyl-6-dehydro-16a,l7a-dihydroxy 19- norprogesterone.

EXAMPLE 2O 6-methyl-6-dehydro-16a,I7ot-(fl-methyl-phenylmethylenedioxy-I 9-norpr0gester0ne To a suspension of6-methyl6-dehydro*16-u,17a-dihydroxy-19-norprogesterone in acetophenoneis added 72% perchloric acid and the mixture is agitated at roomtemperature for three hours. The mixture is then neutralized with dilutesodium bicarbonate and the acetophenone is removed in vacuo. Theresulting crystalline solution is filtered and the crystals washed withwater to yield after recrystallization the 6 methyl 6dehydIO-160l,17OL-(,B- methyl-phenylmethylenedioxy)-19-11orprogesterone.

Similarly, substituting other like acetones or ketones for theacetophenone of Example 20, the respective 16,17- acetal or ketalderivative is obtained.

This invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A compound selected from the group of steroids having the formulawherein P is selected from the group consisting of hydrogen, loweralkyl, halo lower alkyl, monocyclic cycloalkyl, monocyclic cycloalkyllower alkyl, monocyclic aryl,

monocyclic aryl lower alkyl, monocyclic heterocyclic and monocyclicheterocyclic lower alkyl; Q is selected from the group consisting oflower alkyl, halo lower alkyl, monocyclic cycloalkyl, monocycliccycloalkyl lower alkyl, monocyclic aryl, monocyclic aryl lower alkyl,monocyclic heterocyclic and monocyclic heterocyclic lower al-kyl; andtogether with the carbon to which they are joined P and Q is selectedfrom the group consisting of monocyclic cycloalkyl and mono cyclicheterocyclic and Z is selected from the group consisting of a single anddouble bond.

2. 6a methyl 160:,17u dimethylrnethylenedioxy-l9- oxoprogesterone.

3. 6a methyl 1602,1704(fi-methyl-ot-phenylrnethylenedioxy)-19-oxoprogesterone.

4. 6 methyl 6 dehydrol6u,l7a-dimethylrnethylenedioxy-19-oxoprogesterone.

References Cited by the Examiner UNITED STATES PATENTS 3,039,926 6/1962Shull 167-65 3,158,630 11/1964 Cross 260-397.4 3,206,459 9/1965 Cross260-23955 3,234,214 2/1966 Diassi et al. 260239.55

LEWIS GOTT S, Primary Examiner. H. A. FRENCH, Assistant Examiner.

1. A COMPOUND SELECTED FROMTHE GROUP OF STEROIDS HAVING THE FORMULA 